Ampure bead dna purification

SMARCA4 (BRG1) levels of the HA-purified mSWI/SNF complex purifications were normalized via BCA protein quantification and Silver Stain analyses. Purified mSWI/SNF complexes were diluted for final reaction concentration of 10 ng/μL in REAA buffer (20 mM HEPES, pH 8.0, 50 mM KCl, 5 mM MgCl₂) containing 0.1 mg/mL BSA, 1 μM DTT, 20 nM nucleosomes (EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1, EpiCypher). The REAA mixture was incubated at 30 or 37°C for 10 min, and reaction was initiated using 1-2 mM ATP (Ultrapure ATP, Promega) and 0.005 U/μL DpnII Restriction Enzyme (New England Biolabs). The REAA reaction mixture was quenched with 20-24 mM EDTA and placed on ice. Proteinase K (Ambion) was added at (100 μg/mL)_for 30-60 min, followed by either AMPure bead DNA purification and D1000 HS DNA ScreenTape Analysis (Agilent) or mixing with GelPilot Loading Dye (QIAGEN) and loading onto 8% TBE gel (Novex 8% TBE Gels, Thermo Fisher). TBE gels were stained with either SYBR-Safe (Invitrogen) or Syto-60 Red Fluorescent Nucleic Acid Stain (Invitrogen), followed by imaging with UV light on an Alpha Innotech AlphaImager 2200 and/or with 652 nm light excitation on a Li-Cor Odyssey CLx imaging system (LI-COR).

SMARCA4 (BRG1) levels of the HA-purified BAF complex purifications were normalized via BCA protein quantification and immunoblotting analyses. Purified canonical BAF complexes were diluted for final reaction concentration of 10 ng/μL in REAA buffer (20 mM HEPES, pH 8.0, 50 mM KCl, 5 mM MgCl₂) containing 0.1 mg/mL BSA, 1 μM DTT, 20 nM nucleosomes (EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1, EpiCypher). The REAA mixture was incubated at 30 or 37°C for 10 minutes, and the reaction was initiated by addition of 1-2 mM ATP (Ultrapure ATP, Promega) and 0.005 U/μL DpnII Restriction Enzyme (New England Biolabs). The REAA reaction mixture was quenched with 20-24 mM EDTA and placed on ice. Proteinase K (Ambion) was added at (100 μg/mL) for 30-60 minutes, followed by either AMPure bead DNA purification and D1000 HS DNA ScreenTape Analysis (Agilent)